In cartilage, the predominant extracellular matrix molecules synthesized by the chondrocyte are type II collagen, aggrecan, biglycan, decorin and fibromodulin. During the last four years of this grant we have investigated the expression of these ECM molecules by both chondrocytes and other connective tissue cells. Detailed analysis of the COL2A1 gene has revealed a very unique and interesting gene whose expression is complexly regulated both quantitatively at the level of transcription and qualitatively by alternative splicing of exon 2. Interestingly, we and others have found significant expression of the gene in non-cartilage tissue primarily in the embryo. From in situ hybridization studies, we have found high expression of mRNAs in human fetal skeletal tissues, bovine newborn growth plate, in mouse somites, mesenchyme, epithelium and cartilage, and during development of avian long bones and skin. Type II procollagen is "reexpressed" in human osteoarthritis. Studies from our laboratory have shown that the human COL2A1 gene contains many transcriptional regulatory domains in both the promoter and first intron, including a very powerful basic promoter and an intron enhancer. In addition, we have discovered that alternative splicing of the mRNA is tissue-specific with the majority of the non-cartilage molecules containing exon 2 (type IIA) while chondrocytes synthesis mRNA without exon 2 (type IIB). The present proposal will focus on the transcriptional and post-transcriptional regulation of this gene. The specific aims of this proposal are: (1) investigate transcriptional control elements in vitro using transient transfection, "gel-shift" and methylation interference analyses with cell lysates (2) investigate the transcriptional regulation of the COL2A1 gene in vivo using the technology of transgenic mice, and (3) investigate the mechanism of alternative splicing of the COL2A1 gene in vivo and in vitro by in situ hybridization to tissue and transient transfection of splice constructs into cells. The data generated from these studies should be important for the understanding of COL2A1 expression during embryogenesis, skeletal development, chondrogenesis and the neo-synthesis of cartilage during attempts at repair.